Methods and compositions to facilitate repair of avascular tissue

ABSTRACT

Compositions and methods are provided for repairing damaged avascular zones, including intervertebral disc, in a patient in need thereof.

TECHNICAL FIELD

The present invention provides compositions and methods for facilitatingrepair in a damaged avascular site, for example an intervertebral disc;more particularly, the invention provides applying environmentallyconditioned autologous stem cells at optimized locations withinavascular sites or adjacent to avascular sites in patients in needthereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of co-pending U.S.patent application Ser. No. 16/441,897, which is a continuationapplication of U.S. patent application Ser. No. 13/132,840, which is aU.S. national stage application, filed under 35 U.S.C. § 371, ofInternational Application No. PCT/US2009/066773, which was filed on Dec.4, 2009, and which claims priority to U.S. Provisional Application No.61/120,098, which was filed on Dec. 5, 2008 and U.S. ProvisionalApplication 61/154,874, which was filed on Feb. 24, 2009. The contentsof each are incorporated by reference into this specification.

BACKGROUND

Avascular transition zones and other hard to repair sites are present ina number of key tissues of the body. These zones are present where bloodsupply to the tissue, for example a disc, is limited or lacking or wheredamage to the tissue has caused a harsh environment that resists repairprocedures. For example, when an avascular tissue is damaged, the lackor limit of blood supply to that tissue poses a significant hurdle torepair processes. This is particularly true in the intervertebral disc,knee and hip, where normal load issues make it difficult to facilitaterepair and healing.

One particularly important avascular transition zone in the body iswithin the intervertebral disc, where there is no direct blood supply.Nutrients to the disc typically arrive via small capillary beds in thesubchondral bone which diffuse throughout the disc over the course oftime. In addition, discs receive nutrients via imbibitions, in otherwords by soaking up nutrients from surrounding tissue during axialloading activities such as walking, running and the like.

Intervertebral disc are shock absorbing pads that separate any twovertebrae of the spine from one another. These discs essentially providethree functions to a spine, first they act as shock absorbers to carryaxial load of the body while in an upright position, second they act asa ligament to hold any two adjacent vertebrae together, and third theyact as pivot points for enhanced bending and rotation of the spine.

Humans have 23 discs in their spine, i.e., 6 in the cervical region, 12in the thoracic region, and 5 in the lumbar region. Each disc iscomposed of a nucleus pulposus, annulus fibrosus and vertebralend-plates. The nucleus pulposus is water-rich and gelatinous andcomprises the center region of a disc. The annulus fibrosus is fibrousin nature, being made of collagen and includes little water (as comparedto the nucleus pulposus) and surrounds the nucleus pulposus. A series oflamellae are arranged in the annulus fibrosus in order to contain thepressurized nucleus pulposus. In addition, vertebral end-plates act toattach each disc to adjacent vertebral bodies.

As discussed above, tissue repair and regeneration have proven difficultin damaged disc due to harsh environmental aspects of the disc(avascular, high pressure, adverse pH, etc) and the difficult mechanicalrequirements placed on a disc during repair (stress and strainassociated with bipedal movement). Conventional repair methodologiesthat utilize stem cell technology have focused on direct implantation ofstem cells (typically obtained from pre-existing non-autologous celllines) into the nucleus pulposus, typically in the presence of a carriermaterial. While these methodologies have provided some hopeful resultsin animal models, these repairs have yet to be demonstrated in humanswith degenerative disc disease (DDD) or other like conditions. Thepromising results in these animal models are likely due to the acutenature of the disc degeneration model used in animal research. Forexample, the discs in these animal models are newly degenerated with abetter blood supply than a long standing degenerative disc in a human.In addition, the animals used in these models are generally quadrapedalversus humans who are bipedal and as such load discs differently.Finally, the animals used in these DDD models tend to be young andhealthy, equivalent in age to patients who are much younger than thecohort commonly seen clinically with DDD. In general therefore,conventional methodologies are based on repair and regeneration indamaged disc environments potentially very different from those found inhuman patients in need of stem cell therapy.

Issues relevant to tissue repair and regeneration in the disc are alsoprevalent in the hip, knee, and shoulder (including rotator cuff). Ineach of these tissues, harsh environmental aspects are often establishedupon injury or aging, where avascular transition zones and difficultmechanical requirements combine to establish scenarios of low stem cellrepair success.

The present invention is directed toward overcoming one or more of theproblems discussed above.

Summary of the Embodiments

The present invention provides compositions and methods for use inrepair of damaged tissue having one or more poor nutritional zones orhostile environments, i.e., avascular zones. Poor nutritional zones orhostile environments are typically located where the tissue has limitedor lacks vascular blood supply, i.e., termed avascular transition zonesor avascular zones herein. Zones or environments in this light include:intervertebral disc, hip (labrum), shoulder (including rotator cuff) andother like sites. Embodiments herein include procurement of stem cells,for example mesenchymal stem cells, from a patient in need of repair inan avascular zone. Procured cells are then conditioned, in vitro, in anenvironment that allows for optimization of cells capacity to be used inrepair of the patient's avascular zone. When a sufficient number ofoptimized, conditioned cells are present, cells are placed in targetsites of the avascular zone in need of repair. In some embodiments,platelets or platelet lysate (typically autologous) and/or supplementtreatments are placed in combination with the conditioned cells toenhance blood flow/nutrient flow to the site. Timing of the plateletand/or supplement treatment is typically just prior, during or justafter placement of conditioned cells, although other timing regimentsare contemplated. Procedures can be repeated to ensure repair of site,including repeat of only conditioned cell placement or platelet/plateletlysate/supplement treatment(s).

In one embodiment compositions and methods are provided for use inrepair of damaged intervertebral discs in a patient in need thereof. Inone aspect, methods are provided for procurement and culturing of stemcells under conditions which optimize the cells capacity to be used inrepair and/or regeneration of a damaged disc. In another aspect, methodsare provided for placement of these conditioned stem cells in targetedsites of the damaged disc to optimize growth and regeneration of thedamaged disc tissue. In yet another aspect, methods are provided forplacement of the conditioned stem cells in targeted sites within thepatient in combination with treatment to the patient using epiduralsupplement treatment to enhance blood flow to and through the damageddisc. Additionally, the present invention provides improved compositionsfor stem cell culture dedicated toward selection of cells capable ofrepair and/or regeneration of a damaged disc. Individually, and/or incombination, the methods and compositions of the present inventionprovide surprising and unexpected advancements in the field of discrepair and regeneration (as compared to other conventionaltechnologies).

In another embodiment, stem cells (mesenchymal stem cells, for example)are harvested from a patient in need of disc repair and cultured underconditions based on selecting and expanding cells able to withstand apoor nutritional environment, an otherwise hostile environment (forexample one where pH is not in the ranges normally consistent withpromoting healthy cell growth), a hypoxic environment, and/or anenvironment exhibiting elevated carbon dioxide levels within a damageddisc. These conditioned cells are then implanted in the fibrousposterior disc annulus (as compared to conventional methodologies whichtypically call for implantation in the nucleus pulposus). In some casesthe patient is then treated with epidural supplements (growth factors,cytokines, integrins, cadherins, etc.) to facilitate blood flow to theposterior disc annulus. Each aspect of the embodiment increases andselects for stem cells capable of viability and expansion in the damageddisc environment as well as facilitates the disc environment to provideenhanced nutrition and oxygen to the implanted cells. In addition,autologous platelet or platelet lysate compositions can be administeredseparately or in combination with the selected stem cells to facilitatestem cell viability and expansion within the damaged disc. Individuallyor in combination the approaches herein enhance the repair process andresults of autologous stem cell based disc repair.

In some instances, harvested stem cells from the patient in need of discrepair are cultured in vitro under 1-10% oxygen and more typically under3 to 7% oxygen for a period of from 1 to 28 days. This can representapproximately ⅓ to all of the time the cells are cultured prior toimplantation within the patient. Surviving/viable cells, i.e., cellscapable of growth under hypoxic conditions, are selected for viabilityand expanded under these hypoxic conditions to procure enough cells forimplantation into the damaged disc. Selected cells have enhancedcapability to survive, expand and ultimately repair within the oxygendeficient environment of a damaged disc.

In other instances, harvested stem cells from the patient in need ofdisc repair are cultured in vitro under elevated carbon dioxide and moretypically under 2 to 10% carbon dioxide for a period of from 1 to 28days. This can represent approximately ⅓ to all of the time the cellsare cultured prior to implantation within the patient. Surviving/viablecells, i.e., cells capable of growth under elevated carbon dioxideconditions, were selected for viability and expanded under theseconditions to procure enough cells for implantation into the damageddisc. Selected cells have enhanced capability to survive, expand andrepair within the higher carbon dioxide conditions of the environment ofa damaged disc. This same approach can be used to select for stem cellsused to repair/regenerate damaged (through injury or aging) hip and/orshoulder avascular sites.

In other instances, harvested stem cells from the patient in need ofdisc repair are cultured in vitro under both hypoxic and elevated carbondioxide conditions. In vitro culture conditions can be maintained for aperiod of up to ⅓ to all of the total culture time of the cells.Selected cells have enhanced capability to survive, expand and repairwithin the lower oxygen and higher carbon dioxide conditions typicallyfound in damaged intervertebral disc. This same approach can be used toselect for stem cells used to repair/regenerate damaged (through injuryor aging) hip and/or shoulder avascular sites.

In still other instances, harvested stem cells from the patient in needof disc repair are cultured under nutrient poor conditions to select forviability and are expanded under these poor nutrient environments.Culture conditions include use of a basal cell culture media preparedfrom Dulbecco's Modified Essential Medium (DMEM) (or other like basalmedia) supplemented with sugars, amino acids, lipids, minerals,proteins, or other substances intended to facilitate stem cell growth.Growth media may or may not contain serum such as fetal calf serum,human whole serum, platelet rich plasma, platelet lysate, etc. . . . .However, these preparations are specifically designed to mimic the localenvironment of a human degenerated disc such as hypoxia, altered pH, orcertain limited nutrient availability. This same approach can be used toselect for stem cells used to repair/regenerate damaged (through injuryor aging) hip (for example in labrum) and/or shoulder avascular sites.

Other selection conditions can be used to expand harvested stem cellsincluding: pH, use of spent media, co-culture with nucleus pulposuscells, where the target site is in a damaged disc (thereby providing theenvironmental factors present from being in proximity to the ultimatetarget site for delivery of the cultured cells), and the like. Notealso, in some instances two or more of the selection conditionsdescribed herein can be used to identify and expand stem cells for usein avascular site repair. So for instance, poor nutritional media andhypoxic conditions can be used to select for the stem cells used in afirst patient, while pH and carbon dioxide conditions may be used forselection in a second patient. This may be based on actual measurementsof the local micro environment in any given patient.

In still further instances, platelets, from the same patient having thedamaged site, are procured (harvested) and treated with thrombin andcalcium chloride (CaCl₂). Treated platelets are combined with culturedand selected stem cells for implantation into the damaged site. Notethat treatment of the platelets can extend from one to seven days andmore particularly from 5 to 7 days prior to implantation into damagedsite. Additionally, platelets can be implanted just prior to, during orafter implantation of the selected stem cells. Such preconditionedplatelets are capable of releasing targeted growth factors into theavascular zone environment useful in facilitating stem cell survival andexpansion within the damaged site. Alternatively, or in combination,growth factors, cytokines, integrins, etc can be directly administeredwith the selected stem cells into the damaged site. In one embodiment,these growth factors are administered around the exterior of, forexample, a damaged disc such as placed into the epidural space.

These and various other features and advantages of the invention will beapparent from a reading of the following detailed description and areview of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an axial lumbar view with superimposed path of needleplacement of stem cells into the vascular and transitional vascularzones of the posterior disc annulus using embodiments described herein.

FIG. 2 shows MSCs grown in monolayer culture according to a techniqueprovided herein.

FIG. 3 is an exemplary fluoroscopy image of the injection location ofMSCs and platelet derived VEGF supernatant into the posterior discannulus of the L5-S1 disc (subject ML). Note the concentration ofcontrast in the posterior disc annulus (contrast flow enhanced withblue).

FIG. 4 demonstrates exemplary epidural flow attained with the plateletderived VEGF supernatant injections that were performed after the stemcell transplant.

FIG. 5 provides ML Short Tau Inversion Recovery (STIR) image taken lessthan 1 month prior to procedure. This sagittal slice is chosen as itrepresents the maximum extent of the contained L5-S1 disc extrusion.ET=6, TR=4816.7, TE=48.1 with an imaging time of day of 1:01 p.m. Thisimage demonstrates a 0.7 cm disc extrusion at L5-S1. L5-S1 disc heightmeasured at central disc is 0.5 cm with L4-L5 measuring at 0.7 cm.

FIG. 6 provides ML 1 month post procedure matching sagittal slice usingthe same STIR parameters. ET=6, TR=4816.7, TE=48.1. Imaging time of daywas 11:01 a.m. This image demonstrates a 0.3 cm disc extrusion at L5-S1.Note disc heights 0.5 cm at L5-S1 and 0.7 cm at L4-L5.

FIG. 7 provides ML 5 month post procedure matching sagittal slice usingthe same STIR parameters. ET=6, TR=4816.7, TE=48.3. Imaging time of daywas 10:23 a.m. This image demonstrates a 0.3 cm disc extrusion at L5-S1.Note disc heights 0.5 cm at L5-S1 and 0.7 cm at L4-L5.

FIG. 8 provides MJM pre-procedure sagittal slice through the maximumextent of the contained L4-L5 disc extrusion. Image was taken at 12:15pm. ET=6, TR=4816.7, TE=48.1. The L4-L5 disc extrusion is measured at 6mm. Disc heights measured at the mid-portion of the disc on this slicewere: L4-L5=8 mm, L5-S1=7 mm, S1-S2=5 mm.

FIG. 9 provides MJM 2 months post-procedure matching sagittal STIR slicewith same imaging parameters. ET=6, TR=4816.7, TE=48.1. Image was takenat 12:35 pm. The L4-L5 disc extrusion is measured at 3 mm. Disc heightsmeasured the same as pre-procedure: L4-L5=8 mm, L5-S1=7 mm, S1-S2=5 mm.

FIG. 10 provides MJM 4.5 months post-procedure. This is matchingsagittal STIR slice with same imaging parameters. ET=6, TR=4833.3,TE=48.2. Image was taken at 12:27 pm. The L4-L5 disc extrusion ismeasured at 3 mm. Disc heights measured the same as pre-procedure:L4-L5=8 mm, L5-S1=7 mm, S1-S2=5 mm.

FIG. 11 provides HO pre-procedure sagittal STIR slice through themaximum extent of the L5-S1 disc protrusion. ET=6, TR=4816.7, TE=48.3.Image time of day was 11:26 a.m. The L5-S1 disc protrusion is measuredat 9 mm. Disc heights measure: L4-L5=6 mm, L5-S1=8 mm.

FIG. 12 provides HO 6 week post procedure sagittal matching STIR slice.Imaging parameters kept constant at ET=6, TR=4816.7, TE=48.3. Image timeof day was 11:25 a.m. The L5-S1 disc protrusion is measured at 8 mm.Disc heights measure: L4-L5=6 mm, L5-S1=8 mm.

FIG. 13 provides HO 3.5 month post procedure matching sagittal STIRslice. Imaging parameters kept constant at ET=6, TR=4816.7, TE=48.3.Image time of day was 1:10 p.m. The L5-S1 disc protrusion is measured at9 mm. Disc heights measure: L4-L5=6 mm, L5-S1=8 mm.

FIG. 14 shows contrast flow in HO (enhanced in blue) was more typical ofa nucleogram than the intended target which was the posterior discannulus.

DETAILED DESCRIPTION

The present invention provides compositions and methods for use inrepair of damaged tissue having one or more poor nutritional zones orhostile environments, i.e., avascular zones. Poor nutritional zones orhostile environments are typically located where the tissue has limitedor lacks vascular blood supply, i.e., termed avascular transition zonesherein. Zones or environments in this light include: intervertebraldisc, hip, shoulder (including rotator cuff) and other like sites.Embodiments herein include procurement of stem cells, for examplemesenchymal stem cells, from a patient in need of repair in an avascularzone. Procured cells are then conditioned, in vitro, in an environmentthat allows for optimization of cells capacity to be used in repair ofthe patient's avascular zone. When a sufficient number of optimized,conditioned cells are present, cells are placed in target sites of theavascular zone in need of repair. In some embodiments, supplementtreatments are placed in combination with the conditioned cells toenhance blood flow/nutrient flow to the site. Timing of the supplementtreatment is typically just prior, during or just after placement ofconditioned cells, although other timing regiments are contemplated.Procedures can be repeated to ensure repair of site, including repeat ofonly conditioned cell placement or supplement treatment.

Avascular zone conditions for stem cell selection herein generallyinclude: 1-10% oxygen, 2-10% carbon dioxide, altered pH, alterednutrition, and combinations of the like. Selected cells can be placedinto repair sites in combination with supplements, e.g., growth factors,cytokines, integrins, cadherins, and the like, and/or with treatedautologous platelets.

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

“Stem cell(s)” as used herein refers to cells possessing the propertiesof self-renewal and potency. With regard to mesenchymal stem cells,these cells are multipotent and have the capability to differentiateinto osteoblasts, chondrocytes, myocytes, adipocytes, and other likecells.

Disc Bulge as used herein refers to a protrusion of the nucleus pulposisinto the annulus fibrosis of the disc.

Disc Hernation as used herein refers to an extrusion of the nucleuspulposis beyond the confines of the annulus fibrosis.

Contained disc herniation as used herein refers to an extrusion of thenucleus pulposis beyond the confines of the annulus fibrosis and stillconfined by the posterior longitudinal ligament.

“Patient” as used herein refers to a mammal, and more typically a human,having one or more damaged or aged avascular sites, for example damagedintervertebral discs. With regard to a damaged disc, damage may includeherniated disc, bulging disc, fractured disc, disc protrusion, discextrusion, disc sequestration and other like disc ailments.

“Platelet and platelet lysate” are used interchangeably herein andinclude the combination of natural growth factors contained in plateletsthat have been released through lysing of the platelets. This can beaccomplished through chemical means (i.e. CaCl₂), osmotic means (use ofdistilled H₂O), or through freezing/thawing procedures. Platelet lysatesof the invention can also be derived from whole blood and can beprepared as described in U.S. Pat. No. 5,198,357, which is incorporatedby reference herein.

“Repair” or “regeneration” are used interchangeably and refer to partialor complete replacement of a damaged area within a target avascular zoneor a zone adjacent to the avascular zone. For example, repair of anintervertebral disc includes partial or complete repair or replacementof the tissue within the disc. Repair or regeneration can also refer torepair or regeneration of an avascular zone in a normally aging patient,i.e., repair damage to a zone induced by age.

“Environment” as used herein refers to the entire set of conditions thateffect or influence cells in vitro or in vivo.

“Hypoxia or hypoxic” as used herein refers to an in vitro or in vivocondition having 10% or less oxygen in the environment.

“Supplement” or supplement treatments include growth factors, cytokines,integrins, including: VEGF-A, PIGF, VEGF-B, VEGF-C, VEGF-D, FGF, Ang1,Ang2, MCP-1 endoglin, TGF-β, CCL2, VE-cadherin, etc.

Embodiments in accordance with the present invention include methods andcompositions useful in repair and regeneration of damaged avascularzones, e.g., intervertebral discs. Embodiments herein are predicated onthe unexpected finding that harvest and expansion of autologous stemcells under poor nutritional and hypoxic conditions provide more capablecells for repair of an avascular zone. Further, implantation ofconditioned cells with autologous platelets, as well as facilitatingblood flow to the damaged site yield dramatically enhanced repair.Implantation can also include one or more supplement treatment(s) (withor without platelets). Finally, embodiments herein include administeringthese conditioned cells and ancillary materials at optimized siteswithin the damaged site to further facilitate repair and/orregeneration.

Stem Cells

Mesenchymal stem cells (MSCs) hold great promise as therapeutic agentsin regenerative medicine. Alhadlaq, A. and J. J. Mao, Mesenchymal stemcells: isolation and therapeutics. Stem Cells Dev, 2004. 13(4): p.436-48. Barry, F. P., Mesenchymal stem cell therapy in joint disease.Novartis Found Symp, 2003. 249: p. 86-96; discussion 96-102, 170-4,239-41. Bruder, S. P., D. J. Fink, and A. I. Caplan, Mesenchymal stemcells in bone development, bone repair, and skeletal regenerationtherapy. J Cell Biochem, 1994. 56(3): p. 283-94. Cha, J. and V. Falanga,Stem cells in cutaneous wound healing. Clin Dermatol, 2007. 25(1): p.73-8. Gangji, V., M. Toungouz, and J. P. Hauzeur, Stem cell therapy forosteonecrosis of the femoral head. Expert Opin Biol Ther, 2005. 5(4): p.437-42. These adult stem cells can be easily isolated from many sourcesin the body. Alhadlaq, A. and J. J. Mao, Mesenchymal stem cells:isolation and therapeutics. Stem Cells Dev, 2004. 13(4): p. 436-48. Inaddition, they have demonstrated in numerous animal studies, the abilityto differentiate into muscle, bone, cartilage, nerves, tendon, andvarious internal organs cells. Lumbar disc degeneration and pathologyare major causes of significant disability and medical expense.Dagenais, S., J. Caro, and S. Haldeman, A systematic review of low backpain cost of illness studies in the United States and internationally.Spine J, 2008. 8(1): p. 8-20. Surgical treatments such as discectomy,fusion, and disc replacement have been utilized in clinical practice,with strong potential for significant morbidity. de Kleuver, M., F. C.Oner, and W. C. Jacobs, Total disc replacement for chronic low backpain: background and a systematic review of the literature. Eur Spine J,2003. 12(2): p. 108-16. Gotfryd, A. and O. Avanzi, A systematic reviewof randomized clinical trials using posterior discectomy to treat lumbardisc herniations. Int Orthop, 2008. Katonis, P., et al., Postoperativeinfections of the thoracic and lumbar spine: a review of 18 cases. ClinOrthop Relat Res, 2007. 454: p. 114-9. As a result, the ability torepair the Intervertebral disc (IVD) rather than surgical alteration orremoval is an attractive treatment option. Saki and others have shownthat MSC's are capable of lumbar disc repair in animal studies using apuncture model of simulated disc degeneration. Sakai, D., et al.,Regenerative effects of transplanting mesenchymal stem cells embedded inatelocollagen to the degenerated intervertebral disc. Biomaterials,2006. 27(3): p. 335-345. Sakai, D., et al., Differentiation ofmesenchymal stem cells transplanted to a rabbit degenerative disc model:potential and limitations for stem cell therapy in disc regeneration.Spine, 2005. 30(21): p. 2379-87. Sakai, D., et al., Transplantation ofmesenchymal stem cells embedded in Atelocollagen gel to theintervertebral disc: a potential therapeutic model for discdegeneration. Biomaterials, 2003. 24(20): p. 3531-41. The inventorsrecognized that there are many physiologic differences between animaland human IVD models. These include different forces created withquadrupeds in ovine, porcine and murine animals, versus bipedalmechanics in humans. In addition, the popular puncture model ofdegenerative disc disease (DDD), used by many animal researchers createsthe scientific equivalent of an acutely injured disc. Human DDD is oftenpresent for decades prior to the patient seeking medical or surgicaltreatment. Embodiments herein (see Examples) show that MSC'spercutaneously deployed into a posterior disc annulus of human subjectswith VEGF enriched, platelet derived supernatant provides significantdisc repair. The decision to place cells into the posterior disc annuluswas based, partly, on the higher vascularization of this area versus thewell defined avascular, low density nutrient environment within thenucleus pulposus.

Embodiments of the invention include harvest of stem cells from thepatient in need of avascular site repair. Stem cells in accordance withthe invention are as described above in the definitions section. In someembodiments the stem cells are mesenchymal stem cells, i.e., multipotentcells capable of differentiating into, among other cell types,osteoblasts, chondrocytes, myocytes, adipocytes and pancreatic isletcells. Note that for purposes of the invention many embodiments aredescribed in relation to mesenchymal stem cells, although other stemcell types can also be used and are within the scope of the presentinvention.

Stem cell harvest in accordance with aspects of the present inventioninclude those described in US Patent Application S/N PCT/us08/68202which are incorporated by reference in there entirety. Additionally,methods and compositions as described in U.S. Pat. Nos. 5,486,359,6,387,367 and 5,197,985 are incorporated by reference herein in theirentirety.

In more detail, mesenchymal stem cells are multipotent stem cellslocated in the bone marrow, peripheral blood, adipose tissue and otherlike sources. MSCs have the capacity to differentiate into a number ofcell types, including osteoblasts, chondrocytes, myocytes, adipocytes,and beta-pancreatic islet cells.

Source MSCs of the invention are typically harvested from the iliaccrest of the patient in need (or other source such as the IVD,periosteum, synovial fluid, or the vertebral body or pedicle) of therestorative/replacement therapy (or a suitable donor), such patient isreferred to herein as a “patient in need or patient in need thereof”(note that other sources, such as adipose tissue, synovial tissue, andconnective tissue have recently been identified and are also consideredas MSC sources within the scope of the present invention). In oneembodiment, approximately 10-100 cc of bone marrow is harvested and“isolated” using methods described in U.S. Patent Application 60/761,441to Centeno or through adherence to plastic, as described in U.S. Pat.No. 5,486,359 to Caplan et al. Each of these references is incorporatedherein in their entirety for all purposes.

As described in more detail below embodiments of the present inventionmay also require some level or amounts of platelets. As such, thisinvention incorporates changes to standard marrow draw procedures toallow appropriate nucleated cell number yield to use platelets orplatelet lysate techniques. In addition, these platelets can be obtainedfrom whole blood. Since the vast majority of the published research isagain performed in healthy humans or animals, the application of thistechnique to humans with various disease states has never been tested.Note that, the use of an altered technique drawing three small 2-3 ccmarrow aliquots on each side (total of 6 aliquots), produced therequired nucleated cell yield which was successfully expanded in 20%platelet lysate.

Platelets and platelet lysate for use herein is prepared from the bonemarrow harvest using the method of Doucet (Doucet, Ernou et al., 2005 J.Cell Physiol 205(2): 288-36), which is incorporated by reference hereinin its entirety. Typical lysates include from about tens of millions to100's of billions platelets. As shown by Martineau et al., Biomaterials,2004 25(18) p 4489-503 (incorporated herein by reference in itsentirety), platelet lysates inherently include the growth factorsrequired to facilitate consistent MSC growth. In typical embodiments theplatelet lysate and MSC are autologous and are in amounts useful foreffective and consistent expansion of the MSCs (described more fullybelow). In particular, it should be noted that while the levels ofgrowth factors such as TGF-beta are much lower in platelet lysate thanthose commonly used to expand MSC's, it is believed that there aresignificant synergistic effects when all of the low level growth factorscontained in platelet lysate are used together.

Stem Cell Selection (Selective Pressure)

Harvested stem cells are cultured to select for stem cells (typicallymesenchymal stem cells) and ultimately for stem cells that are viableand expand under environmental conditions similar to those conditionsfound in a site in need of repair, for example a disc in need of repair.Selective pressure as it relates to intervertebral disc repair isdiscussed in greater detail below, but similar conditions are presentand ascertainable for conditions required for hip, shoulder and thelike.

As discussed in Urban et al., Nutrition of the intervertebral Disc.Spine, 2004. 29 (23): p 2700-9, (incorporated herein by reference in itsentirety), intervertebral disc that have suffered injury and aredegenerative provide a poor nutritional as well as oxygen environment.This environment is distinct from the environment of a healthyintervertebral disc. In fact, studies performed to determine viabilityof transplanted mesenchymal stem cells in injured disc show poor cellviability results, with few cells capable of expanding to provide thenecessary numbers of cells needed for enhanced disc repair (Wuertz etal., Behavior of mesenchymal stem cells in the chemical microenvironmentof the intervertebral disc. Spine, 2008. 33(17): p 1843-9, incorporatedherein by reference in its entirety).

In more detail, harvested stem cells from a patient in need of discrepair or restoration are placed under culture conditions. In oneembodiment, the culture medium is a basal cell culture medium preparedfrom DMEM or other like media. Culture medium can be supplemented withsugars, amino acids, lipids, minerals, proteins, or other likesubstances intended to facilitate stem cell expansion.

In addition, embodiments herein can include culturing harvested andexpanding mesenchymal stem cells under various atmospheric conditionsthat simulate a damaged disc's environment. In one embodiment, harvestedstem cells are cultured in vitro under 1-15% oxygen. In some cases theharvested stem cells are cultured under 3 to 10% oxygen and in othercases the harvested stem cells are cultured under 3 to 7% oxygen. Theselower oxygen conditions replicate the hypoxic conditions present intypical damaged disc environments.

Hypoxic conditions can be present for part or all of the stem cellexpansion period but is typically present for at least ⅓ of the timethat cells are cultured in vitro. Selection occurs as cells arecultured, with viable cells that are able to survive and ultimatelyexpand having an advantage when implanted into a disc having a hypoxicenvironment.

In other embodiments, harvested stem cells are cultured in vitro underelevated carbon dioxide conditions, typically from 2-10% carbon dioxide.Harvested cells can additionally be cultured in a combined elevatedcarbon dioxide and hypoxic environment, where conditions include from2-10% carbon dioxide and from 3-10% oxygen. As above, selection occursas cells are cultured, with viable cells that are able to survive andultimately expand having an advantage when implanted into a disc havingan elevated carbon dioxide environment or an elevated carbon dioxideenvironment combined with a hypoxic environment.

In other embodiments, harvested stem cells are cultured and expanded incombination with harvested and cultured nucleas pulposis cells (NPcells) or annulus fibrosis cells (AF cells). The nucleas pulposis (NP)cells for co-culture with stem cells can be harvested from the patientin need of disc repair via a needle aspirate or other like technique.These NP cells or AF cells can be either autologous or non-autologous.In typical embodiments, approximately 10³ to 10⁹ NP or AF cells areco-cultured with the harvested stem cells and are allowed to provide anenvironment useful for selection of stem cells that respond to NP celland/or AF cell released factors and waste products. Co-cultureconditions can include poor nutritional environment, hypoxia, elevatedcarbon dioxide and other disclosed embodiments described herein. In someembodiments, the NP cells are cultured in a separate in vitro flask (orother like container) from the stem cells. The spent media from the NPcell culture can then be combined with media conditions above duringstem cell culture or can be used exclusively to expand and select forstem cells able to maintain viability and ultimately expand under suchconditions.

In other embodiments, harvested stem cells are cultured and expandedunder modified pH conditions similar to those found in a damagedintervertebral disc. For example, in vitro culture media (as describedherein) can be modified to have a pH of from 6.6-7.0, and more typicallyfrom 6.7 to 6.9. Modified pH can be combined with any of the cultureconditions discussed herein to facilitate selection of stem cells foruse in disc repair and regeneration.

In other embodiments, harvested stem cells are cultured and expandedunder modified osmolarity conditions similar to those found in a damagedintervertebral disc. For example, in vitro culture media (as describedherein) can be modified to have an osmolarity of from 350-600 mOsm, andmore typically from 450 to 500 MOsm. Modified osmolarity can be combinedwith any of the culture conditions discussed herein to facilitateselection of stem cells for use in disc repair and regeneration.

In other embodiments, viability and expansion of stem cells under one ormore selection conditions can be modified by inclusion of one or moregrowth factors. In these cases, cells under selection are cultured inthe presence of TGF-beta FGF, PDGF, IGF and/or HIF-1 alpha, includingmixtures thereof and other like factors.

Note that for each of the above stem cell culture based embodiments, thecondition(s) can be gradually incorporated into cells standard cultureenvironment. For example, harvested stem cells may be initially culturedunder 10% oxygen for one or two passages, then moved to 9% oxygenconditions for one or two passages, and cultured under decreasing levelsof oxygen until a target hypoxic condition is obtained. Under thisprocedure, cells are gradually allowed to adapt to an environmentpresent in a damaged disc.

The following treatment procedure is described in relation to treatmentof a damaged disc, although treatment of other avascular zones areenvisioned to be within the scope of the present invention.

Treatment of Damaged Intervertebral Disc

Stem cells having been selected for by at least one of the abovediscussed damaged disc modifiers are allowed to expand until asufficient number of cells are present for implantation into a patient'sdamaged disc. In typical embodiments, from about 10⁵ to 10⁹ selectedmesenchymal stem cells are required for implantation into the damageddisc.

Cultured cells are washed using PBS or other like buffer to obtain acell population that does not include materials not intended forimplantation into the patient's body, i.e., media constituents, wasteproducts, etc. Washed stem cells can include NP cells, although it iscontemplated that where stem cells are co-cultured with NP cells, thatthe NP cell population can be removed via cell sorting techniques oraffinity chromatography. Stem cells are now ready for implantation intothe patient in need thereof.

In one embodiment, the washed stem cells are implanted directly into theposterior annulus of the damaged disc. This is an unexpected locationfor implantation of stem cells, as conventional methodologies showimplantation of cells into the nucleas pulposis. Cells are implantedinto the posterior annulus of the damaged disc via known techniques inthe art, including via percutaneous x-ray guided or surgical IVD access.One or more iteration of cell implantation can be used in repairprocedure for a damaged disc, although, a period of 14 to 180 days istypical between treatments.

In another embodiment, autologous platelets from the patient in need oftherapy are pretreated with thrombin and CaCl₂ for approximately one toseven days. This treatment preconditions these platelets topreferentially express vascular endothelial growth factor (VEGF). Insome embodiments, the harvested platelets are pretreated withapproximately 28.56 U/ml thrombin and approximately 2.86 mg/ml CaCl₂. Inadditional embodiments, autologous platelets from the patient in needthereof are pretreated with a combination of thrombin, calcium or its'salts, thromboxane A2, adenosine triphosphate, and arichidonate. Asabove, pretreatment can be from one to seven days prior to implantationinto the patient in need thereof. The preconditioned autologousplatelets are then implanted before, during or after implantation of thepreviously discussed selected conditioned stem cells of the invention.Platelets are generally implanted in the same location as the implantedstem cells.

In another embodiment, harvested and selected stem cells of theinvention are implanted with one or more supplement or supplementtreatments, including growth factors, cytokines, integrins, cadherins,or molecules or drugs known to promote angiogenesis, vasculogenesis oraerteriogenesis, including: VEGF-A, PIGF, VEGF-B, VEGF-C, VEGF-D, TGF-β,Ang-1, Ang-2, IGF, HGF, FGF, Tie2, PDGF, CCL2, Alpha-V Beta-5, Alpha-5Beta-1, VE-cadherin, PECAM-1, plasminogen activator and nitrogen oxidesynthase. In alternative embodiments, stem cells for use in implantationin patients in need thereof are co-implanted with a combination ofgrowth factors including TGF-β, FGF, PDGF, IGF or other like growthfactors intended to promote stem cell and/or mesenchymal stem cellstemness or proliferation. In some embodiments autologous platelets orplatelet lysates can be implanted in combination with the beforementioned supplements.

FIG. 1 illustrates the utility of one embodiment of the inventionshowing an axial lumbar view with a superimposed needle placement.Conditioned stem cells using embodiments described herein were placed inthe vascular and transition vascular zones of the posterior discannulus.

Examples Example 1: Percutaneously Implanted Autologous Mesenchymal StemCells Methods: Subjects:

Three patient subjects were selected based on willingness to participatein an IRB (Spinal Injury Foundation, Westminster, Colo.) approved MSCimplantation protocol. Each subject signed an IRB approved consent form.Subjects were selected based on the following inclusion/exclusioncriterion:

Inclusion Criteria:

-   -   1. 18-65 years of age    -   2. Failure of conservative management    -   3. Lumbar degenerative disc disease with a disc protrusion or        contained disc extrusion (subligamentous)    -   4. Selective nerve root blocks that confirmed the disc        protrusion/nerve to be treated as the pain generator (>75%        relief of major pain complaint) or discography which confirmed        the disc as a P2 pain generator    -   5. At least 75% of normal disc height with or without        dehydration on T2 weighted MRI images    -   6. Unwillingness to pursue surgical options

Exclusion Criteria:

-   -   1. Active inflammatory or connective tissue disease (i.e. lupus,        fibromyalgia, RA)    -   2. Active non-corrected endocrine disorder potentially        associated with symptoms (i.e. hypothyroidism, diabetes)    -   3. Active neurologic disorder potentially associated with        symptoms (i.e. peripheral neuropathy, multiple sclerosis)    -   4. Severe cardiac disease    -   5. Pulmonary disease requiring medication usage    -   6. A history of dyspnea or other reactions to transfusion of        autologous blood products

Pre-Procedure Data Collection:

1. CBC and blood chemistries were obtained within 3 months of the MSCimplantation to rule out unknown medical conditions.

2. Pre-procedure MRI

3. Pre-procedure outcomes measures

Isolation and Expansion of Mesenchymal Stem Cells (MSCs):

For one week prior to the marrow harvest procedure the patient wasrestricted from taking corticosteroids or NSAIDs. Coincident with themarrow harvest procedure, heparinized (Abraxis Pharmaceuticals) IVvenous blood was drawn to be used for platelet lysate (PL). Plateletlysate was prepared via centrifugation at 200 g to separate plateletrich plasma (PRP) from red blood cells (RBCs). PRP volume was aliquotedand stored at −20° C. to produce PL. Platelet lysate was supplemented incell culture media at 10-20%.

A platelet derived VEGF rich supernatant was also prepared based on themethod described by Martineau. (Martineau, I., E. Lacoste, and G.Gagnon, Effects of calcium and thrombin on growth factor release fromplatelet concentrates: kinetics and regulation of endothelial cellproliferation. Biomaterials, 2004. 25(18): p. 4489-502). Using the samePRP isolation steps as above, the PRP was drawn off and an aliquot wasactivated with 28.56 U/mL of human thrombin (Johnson and Johnson) and2.85 mg/mL of Calcium Chloride (CaCl₂, American Regent) for 6 days at37° C. and 5% CO₂. Activated PRP samples were centrifuged at 3,000 rpmfor 6 minutes, supernatant was draw off and stored at −80° C. This waslater used as both injectate to be mixed with culture expanded MSC's aswell as for supplement injections to be delivered via epiduralinjection.

Coincident with the whole IV blood draw, the patient was then placedprone on an operating room (OR) table and the area to be harvested wasanesthetized with 1% Lidocaine, and a sterile disposable trocar was usedto draw 10 cc of marrow blood from the right PSIS area and 10 cc fromthe left PSIS area, in heparinized syringes. If the patient reportedpain during the marrow draw that was not easily controlled by localanesthetics, a caudal epidural with anesthetic only was added.

Whole marrow was centrifuged at 200 g for 4-6 minutes to separate thenucleated cells from the RBCs. The nucleated cells were removed andplaced in a separate tube. Samples were centrifuged at 1000 g for 6minutes to pellet. The nucleated cells were washed once in phosphatebuffered saline (PBS, GibCo), counted, and then re-suspended inDulbecco's modified eagle medium (DMEM, GibCo) with 10-20% PL, 5 ug/mLdoxycyline (Bedford Labs), and 2 IU/mL heparin (AbraxisPharmaceuticals). Nucleated cells were seeded at 1×10⁶ cells/cm² in atissue culture flask. Cultures were incubated at 37° C./5% CO₂/5% O₂ ina humidified environment. The culture medium was changed after 48-72hours, removing the majority of the non-adherent cell population. TheMSC colonies developed in 6-12 days and then were harvested with animalorigin-free trypsin like enzyme (TrypLE Select, GibCo) such that onlythe colony-forming MSCs detached. To expand the MSCs, they were platedat a density between 6-12,000 cells/cm² in alpha-modified eagle medium(AMEM, GibCo) with 10-20% PL, 5 ug/mL doxycyline, and 2 IU/mL ofheparin, and grown to near confluence at 37° C./5% CO₂/5% O₂. Primarycells derived from the bone marrow were designated as passage 0 and eachsubsequent subculture of MSCs was considered one further passage. SeeFIG. 2 for an example of the MSC morphology grown with this monolayercell culture technique. After MSC's had been sub cultured to the 2nd-5thpassage, they were harvested, washed, and suspended in the activated PRPfor injection.

Percutaneous Implantation Procedure

Subjects were positioned prone on an x-ray table and prepped usingbetadine swab with sterile gloves and drapes. An AP fluoroscopy view(Siemens Iso-C) was obtained with an ipsilateral oblique orientation.The superior endplate of the targeted level to be injected wasvisualized “on end” by adjusting cephalic-caudal tilt. Using steriletechnique, a 22 gauge 7 inch quinke needle was guided under bi-planarfluoroscopy to the superior articular process of the lumbar facet jointof the level to be treated and advanced past the facet into theposterior disc. Once the disc access was obtained, under lateral view,the needle was positioned to maximize contrast flow (Omnipaque 300mg/ml-NDC 0407-1413-51 diluted 50% with PBS) into the posterior annulusas close to the anatomic position of the disc protrusion as possible,using pre-treatment MM imaging for approximation (see FIG. 3). Cultureexpanded autologous MSC's in platelet derived VEGF supernatant were theninjected and the needle was extracted.

Following MSC implantation, at week 1 and week 2 the subject returnedfor additional transformainal epidurals performed with VEGF derivedsupernatant at the target levels. Epidural procedures were performedwith the same preparation. Epidural access was obtained using a 25 gauge3.5 inch quince needle which was manipulated under biplanar fluoroscopyand directed toward the subpedicular recess of the target level beinginjected. Once good epidural dye flow extending upwards and under thepedicle was visualized, the VEGF derived supernatant plus 4% lidocainewas injected and the needle was extracted (see FIG. 4). Post-optreatment protocol consisted of lumbar traction in physical therapy orat home at 3 times a week for four weeks.

Imaging and Patient Follow-Up:

A GE 3.0 Tesla Excite HD was used to image the lumbar spine. Imagingsequences included a sagittal Short Tau Inversion Recovery (STIR), FastGradient Recall Echo (FGRE) sagittal, and a Gradient Recall Echo FastSpin (GRE-FS). Images were viewed and measured in E-Film WorkstationVersion 1.5.3 (Merge Healthcare). Sagittal short tau inversion recovery(STIR) and gradient recall echo (GRE) fast spin images with matchingTR/TE and matching imaging planes were used. This was performed toreduce the likelihood of interpretation error of serial images in thesame patient. To reduce diurnal effects, the imaging center wasinstructed to perform the serial films as close to the same time of dayas possible.

Follow-up questionnaires were initiated via phone and obtained from thepatient concerning function and symptoms. Modified VAS scores wereobtained with regard to low back pain and a “Functional Rating Index”(FM) was also obtained. This questionnaire focuses on patient function.(Feise, R. J. and J. Michael Menke, Functional rating index: a new validand reliable instrument to measure the magnitude of clinical change inspinal conditions. Spine, 2001. 26(1): p. 78-86; discussion 87). Officevisits were also used for post-op examinations. These were initiated atthe following intervals:

1. At 6 weeks post procedure.

2. At 12 weeks post procedure

Results:

Medical histories and outcomes of the three subjects enrolled aredescribed below:

Patient 1:

HO was a 19 year old white female, college athlete with a 7 year historyof low back pain thought to be secondary to lifting trauma and herchosen sport. She had undergone extensive conservative care for fouryears prior to presentation, including evaluation and treatment with aninterventional pain management physician The lumbar facets were ruledout as pain generators with negative response to intra-articularinjections., Mill showed an L5-S1 disc protrusion abutting both 51 nerveroots, worse on the right side. Discography revealed a symptomaticposterior annular tear with concordant reproduction of pain at the L5-51disc. Pre-procedure the subject described constant pain of variableseverity, with numbness and tingling in the 51 distribution of the rightfoot with any physical activity, resulting in significant functionallimitations.

The subject reported up to 75% improvement through 12 weeks. At 6 monthspost procedures, the subject showed little change in her reportedmodified VAS pain scores and Functional Rating Index. There was acorresponding lack of change seen in the post-treatment Mill imagingresults. A 1 mm reduction in maximum protrusion size was seen at 6weeks, but this returned to pre-procedure size at 3.5 months. All discheight measurements remained the same across all imaging sessions andthe maximum time of day interval for all images taken was 1 hour and 45minutes. Results for the HO are shown in FIGS. 11, 12 and 13.

Patient 2:

MJM was a 35 year old white male with a 15 year history of low back painprior to presentation. He reported failed conservative care andincreasing frequency of severe pain episodes due to activity. MMrevealed a lumbarized S1-S2 segment, a broad-based disc bulge withmoderate facet encroachment and moderate foraminal narrowing at L5-S1.Additionally, there was a right greater than left posterior containedprotrusion at L4-L5. The physical examination revealed no activeradiculopathy, but the patient reported intermittent radicular symptomsassociated with exacerbations. Surgery was recommended after failure onconservative management, but was declined. The L4-L5 disc was the focusof this treatment with the dehydrated L5-51 disc left as a control.

Six months post procedure, the patient improvement of modified VAS from3 to a 0 with a drop in frequency of pain by more than 80% (see Table1). The FM score for this patient increased by more than 60% and hereported overall symptom improvement at 50%. L4-L5 disc bulge sagittalSTIR image measured 6 mm at its maximum extent in the pre-op MR images(see FIG. 8). In both the 2 month and 4.5 month follow-up films(matching image sequence and slice-see FIGS. 9 and 10) the L4-L5 bulgewas found to be reduced to 3 mm with no change in disc heights measuredat any of the L4-S2 discs. The time of day when the images were acquiredvaried by no more than 20 minutes.

Patient 3:

ML was a 24 year old white female with a traumatic low back injuryassociated with a military training exercise. At the time ofpresentation she had been symptomatic for three years. Her complaintsconsisted of electric shooting pain down one and sometimes both legs,low back pain with prolonged sitting or standing, and pain with bendingor stooping. She had failed conservative management consisting ofphysical therapy. The pre-procedure MM demonstrated decreased discheight but somewhat preserved T2 signal in the L5-S1 disc (see FIG. 5).This disc also had an extrusion of 0.7 cm that was contained by theposterior longitudinal ligament. The posterior annulus was disrupted andthere was a high intensity zone from the nucleus pulposis to theposterior longitudinal ligament. The L4-L5 disc had less T2 signal andwas dehydrated, but had preservation of disc height. There was aprotrusion at this level measuring 0.4 cm with a high intensity zoneseen in the inferomedial portion of the disc. Based on the history,examination and MM findings, intermittent traversing nerve rootirritation at L5 and S1 roots was suspected at both L4-L5 and L5-S1 disclevels. Discogram was performed showing low pressure concordant pain(P2) at both L4-L5 and L5-S1, both with posterior annular tears. Thedecision was made to treat only the L5-S1 disc with the MSC injection,leaving L4-L5 to serve as an untreated control.

At 6 months post procedure (see Table 1) ML reported improvement ofapproximately 40% in low back modified VAS (1-10 scale) with a decreaseof greater than 60% in frequency of pain. Function improved as measuredby FRI by more than 70%. She self reported symptom improvement of 60%.Her post-procedure lumbar MRI imaging demonstrated that the size of theL5-S1 disc protrusion decreased from 0.7 cm pre-procedure to 0.3 cm at 1month post procedure and 0.4 cm at 5 months post procedure (see FIGS. 6and 7). Additionally, the size of the HIZ area in the posterior discannulus decreased in both follow-up films. The time of image acquisitiondiffered by a no more than 1.5 hours.

TABLE 1 Pre-procedure and 6 month post procedure reported outcomes forlow back modified VAS and frequency. Frequency of 1.0 equals constantpain. Functional Rating Index measurements as well as self report ofpercentage change in condition. VAS- VAS- VAS- VAS- Self- Sub- pre postpre AVG post AVG FRI- FRI- Reported ject AVG AVG frequency frequency prepost Outcome ML 4 2 1.00 0.38 44 13 60% improvement MJM 3 1 1.00 0.13 239 50% improvement HO 7 4 1.0 1.0 27 25 No lasting improvement

TABLE 2 Total MSC yields and time in culture. Final MSC Yield Subject inMillions Days in Culture ML 14.3 18 MJM 33.0 17 HO 28.5 17

Discussion:

Two of the three treated subjects demonstrated a decrease in the size ofthe treated disc protrusion and reported sustained subjective andfunctional improvement. A single subject demonstrated a temporarydecrease in symptoms and a small transient change in the size of thedisc protrusion, followed by regression to pretreatment baselinemeasures. Of interest, placing cells preferentially into the posteriordisc annulus of this patient was technically difficult, with sub-optimalflow of contrast into the posterior annulus. The inventors questionpossible correlation between this suboptimal placement and the subject'slack of sustained response. Koga et al. has demonstrated that MSC'sinjected nonspecifically into the intra-articular space failed to repaircartilage lesions, but those applied directly on the defect, allowingattachment at the target tissue were capable of repair. Note that HO'scontrast flow and subsequent MSC flow (see FIG. 14) was primarily intothe nucleus pulposus. Our own unpublished clinical experience in placingmesenchymal stem cells directly into the nucleus pulposus of otherpatients failed to initiate any observable MRI changes. Theseobservations would also support the concept that location of stem celladherence may be critical to the success of treatment. In particular,the posterior disc annulus maintains some vascular perfusion, while theavascular nucleus pulposus has a well documented suboptimal nutritionalenvironment, which may result in a lack of sustainability of implantedMSC's. Martin, M. D., C. M. Boxell, and D. G. Malone, Pathophysiology oflumbar disc degeneration: a review of the literature. Neurosurg Focus,2002. 13(2): p. E1. Also of note, HO had the least prolific stem cellyield, producing significantly fewer cells over a longer culture periodthan the other subjects. (see Table 2).

Since MSC's have a fibroblastic morphology in monolayer culture (seeFIG. 2) the observed results could have been due to fibroblasticdifferentiation of the MSC's placed preferentially into the posteriordisc annulus. Awad, H. A., et al., In vitro characterization ofmesenchymal stem cell-seeded collagen scaffolds for tendon repair:effects of initial seeding density on contraction kinetics. J BiomedMater Res, 2000. 51(2): p. 233-40. Delorme, B. and P. Charbord, Cultureand characterization of human bone marrow. Mesenchymal stem cells.Methods Mol Med, 2007. 140: p. 67-81. Xiang, Y., et al., Ex vivoexpansion and pluripotential differentiation of cryopreserved human bonemarrow mesenchymal stem cells. J Zhejiang Univ Sci B, 2007. 8(2): p.136-46.

Alternatively, the other variable which may have contributed to atherapeutic effect was the follow up platelet supernatant epiduralinjections. Martineau et al. has shown that a platelet supernatantprepared using specific calcium and thrombin preconditioning, produces amaximal burst of VEGF degranulation from platelets (as well as a host ofother growth factors). Martineau, I., E. Lacoste, and G. Gagnon, Effectsof calcium and thrombin on growth factor release from plateletconcentrates: kinetics and regulation of endothelial cell proliferation.Biomaterials, 2004. 25(18): p. 4489-502. VEGF is known to causeangiogenesis and the human degenerative intervertebral disc (IVD) isknown to suffer from poor vascular perfusion. Maroudas, A., et al.,Factors involved in the nutrition of the human lumbar intervertebraldisc: cellularity and diffusion of glucose in vitro. J Anat, 1975.120(Pt 1): p. 113-30. Wallace, A. L., et al., Humoral regulation ofblood flow in the vertebral endplate. Spine, 1994. 19(12): p. 1324-8.Pandya, N. M., N. S. Dhalla, and D. D. Santani, Angiogenesis—a newtarget for future therapy. Vascul Pharmacol, 2006. 44(5): p. 265-74.

An alternative explanation for the decrease in IVD protrusion in thesepatients may have been attributed to diurnal effects. Park et al.demonstrated changes in the size of disc bulges in 8 asymptomaticvolunteers at L4-L5 when MRIs were performed in the morning and evening.Park, C. O., Diurnal variation in lumbar MRI. Correlation between signalintensity, disc height, and disc bulge. Yonsei Med J, 1997. 38(1): p.8-18. While this effect could have occurred in these subjects, two ofthe patients had untreated control discs that did not change in size. Inaddition, the single imaging center responsible for the MRI studies wasinstructed to perform follow-up scans as close as possible to the sametime of day as the initial scans. As a result, the maximum time of dayvariance between images for any patient was less than 2 hours. Finally,treated and control disc heights were measured and no significantchanges were noted for all subjects. If diurnal changes had beenpresent, one would expect significant variation in disc height due tothe effects of imbibition. An alternative explanation for reduction indisc protrusion size could be a healing effect induced directly byneedle trauma. Korecki et al. investigated this effect in an in-vitroanimal model and found the opposite to be true, needle puncture likelycaused biomechanical injury to the disc and a reduction in its overallfunction. Korecki, C. L., J. J. Costi, and J. C. Iatridis, Needlepuncture injury affects intervertebral disc mechanics and biology in anorgan culture model. Spine, 2008. 33(3): p. 235-41. Additionally, itshould be noted that the standard model for inducing DDD in animals isvia puncture injury. Niinimaki, J., et al., Quantitative magneticresonance imaging of experimentally injured porcine intervertebral disc.Acta Radiol, 2007. 48(6): p. 643-9.

CONCLUSIONS

This Example demonstrates that percutaneously implanted autologous MSC'swith platelet supernatant epidural supplementation is capable ofreducing the size of contained lumbar disc protrusion. Although it isunclear why one of the patients did not respond to the therapy, it isreasonable to hypothesize that lack of response was attributable tocomparatively low MSC yield, as well as the sub-optimal placement of theMSCs.

The data in this Example shows the utility of one embodiment of theinvention where procedures described herein provide a surprisingimprovement in repair over other procedures described in the art. Datawith regard to other avascular repair sites, e.g., shoulder, hip, etc,is expected to show similar levels of improvement.

Various embodiments of the disclosure could also include permutations ofthe various elements recited in the claims as if each dependent claimwas multiple dependent claim incorporating the limitations of each ofthe preceding dependent claims as well as the independent claims. Suchpermutations are expressly within the scope of this disclosure.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

The description of the present invention has been presented for purposesof illustration and description, but is not intended to be exhaustive orlimiting of the invention to the form disclosed. The scope of thepresent invention is limited only by the scope of the following claims.Many modifications and variations will be apparent to those of ordinaryskill in the art. The embodiment described and shown in the figures waschosen and described in order to best explain the principles of theinvention, the practical application, and to enable others of ordinaryskill in the art to understand the invention for various embodimentswith various modifications as are suited to the particular usecontemplated.

This specification contains numerous citations to patent, patentapplication, and publications. Each is hereby incorporated by referencefor all purposes.

What is claimed is:
 1. A pharmaceutical composition comprising:mesenchymal stem cells cultured under selective pressure of about 1% toabout 10% oxygen; a growth factor; and a pharmaceutical carrier ordiluent.
 2. The pharmaceutical composition of claim 1 wherein the growthfactor is selected from the group consisting of TGF-β, FGF, PDGF, IGF,VEGF-B, and combinations thereof.
 3. The pharmaceutical composition ofclaim 1 wherein the growth factor is PDGF.
 4. The pharmaceuticalcomposition of claim 1 wherein the growth factor is FGF.
 5. Thepharmaceutical composition of claim 1 wherein the growth factor isVEGF-B.
 6. The pharmaceutical composition of claim 1 wherein the growthfactor is TGF-β.
 7. The pharmaceutical composition of claim 1 whereinthe mesenchymal stem cells are cultured under selective pressure ofabout 1% to about 10% and from about 2 to about 10% carbon dioxide. 8.The pharmaceutical composition of claim 1 wherein the mesenchymal stemcells are cultured under selective pressure of about 3% to about 7%oxygen.
 9. The pharmaceutical composition of claim 8 wherein themesenchymal stem cells are cultured under selective pressure of about 3%to about 7% and from about 2 to about 10% carbon dioxide.
 10. Apharmaceutical composition comprising: mesenchymal stem cells culturedunder selective pressure of about 1% to about 10% oxygen; platelets; agrowth factor; and a pharmaceutical carrier or diluent.
 11. Thepharmaceutical composition of claim 10 wherein the platelets are treatedwith thrombin and calcium chloride for 1-7 days.
 12. The pharmaceuticalcomposition of claim 10 wherein the platelets are treated with thrombin,calcium chloride or its salts, thromboxane A2, adenosine triphosphateand arachidonate.
 13. The pharmaceutical composition of claim 10 whereinthe growth factor is PDGF.
 14. The pharmaceutical composition of claim10 wherein the growth factor is FGF.
 15. The pharmaceutical compositionof claim 10 wherein the growth factor is VEGF-B.
 16. The pharmaceuticalcomposition of claim 10 wherein the growth factor is TGF-β.
 17. Thepharmaceutical composition of claim 10 wherein the mesenchymal stemcells are cultured under selective pressure of about 1% to about 10% andfrom about 2 to about 10% carbon dioxide.
 18. A pharmaceuticalcomposition comprising: mesenchymal stem cells cultured under selectivepressure of about 1% to about 10% oxygen; a compound; and apharmaceutical carrier or diluent.
 19. The pharmaceutical composition ofclaim 18, wherein the compound is selected from the group consisting ofgrowth factors, cytokines, integrins, cadherins, molecules or drugs thatpromote angiogenesis, molecules or drugs that promote vasculogenesis,and molecules or drugs that promote arteriogenesis.
 20. Thepharmaceutical composition of claim 18 wherein the compound is selectedfrom the group consisting of VEGF-A, PIGF, VEGF-B, VEGF-C, VEGF-D,TGF-β, Ang-1, Ang-2, IGF, HGF, FGF, Tie2, PDGF, CCL2, Alpha-V Beta-5,Alpha-5 Beta-1, VE-cadherin, PECAM-1, plasminogen activator, nitrogenoxide synthase, and combinations thereof.